Clostridial toxin derivatives and methods for treating pain

ABSTRACT

Agents for treating pain, methods for producing the agents and methods for treating pain by administration to a patient of a therapeutically effective amount of the agent. The agent can include a clostridial neurotoxin, or a component or fragment or derivative thereof, attached to a targeting moiety, wherein the targeting moiety is selected from a group consisting of transmission compounds which can be released from neurons upon the transmission of pain signals by the neurons, and compounds substantially similar to the transmission compounds.

CROSS REFERENCE TO RELATED APPLICATION

[0001] This application is a continuation-in-part application of U.S.Ser. No. 09/625,098, filed Jul. 25, 2000, which is acontinuation-in-part application of U.S. Ser. No. 09/489,667, filed Jan.19, 2000, the contents of which in their entireties are incorporated byreference into the present application.

BACKGROUND

[0002] The present invention relates to compositions and methods fortreating pain, including bone tumor pain. In particular, the presentinvention relates to Clostridial toxin derivatives, methods for makingthe Clostridial toxin derivatives, and methods for treating pain usingthe Clostridial toxin derivatives.

[0003] Many, if not most, aliments of the body cause pain. The causes ofpain can include inflammation, muscle spasm and the onset of aneuropathic event or syndrome. Inflammatory pain can occur when tissueis damaged, as can result from surgery or due to an adverse physical,chemical, or thermal event or to infection by a biologic agent.Spasticity or muscle spasm can be a serious complication of trauma tothe spinal cord or other disorders that create damage within the spinalcord. Muscle spasm is often accompanied by pain. The pain experiencedduring a muscle spasm can result from the direct effect of the musclespasm stimulating mechanosensitive pain receptors or from the indirecteffect of the spasm compressing blood vessels and causing ischemia.Since the spasm increases the rate of metabolism in the affected muscletissue, the relative ischemia becomes greater creating therebyconditions for the release of pain inducing substances. Neuropathic painis a persistent or chronic pain syndrome that can result from damage tothe nervous system, the peripheral nerves, the dorsal root ganglion ordorsal root, or to the central nervous system.

[0004] Neuropathic pain syndromes include allodynia, various neuralgiassuch as post herpetic neuralgia and trigeminal neuralgia, phantom pain,and complex regional pain syndromes, such as reflex sympatheticdystrophy and causalgia. Causalgia is characterized by spontaneousburning pain combined with hyperalgesia and allodynia.

[0005] Pain can be experienced when the free nerve endings whichconstitute the pain receptors in the skin as well as in certain internaltissues are subjected to mechanical, thermal, or chemical stimuli. Thepain receptors transmit signals along afferent neurons into the centralnervous system and thence to the brain.

[0006] The transduction of sensory signals, such as pain signals, fromthe periphery to sensation itself is achieved by a multi-neuronalpathway and the information processing centers of the brain. The firstnerve cells of the pathway involved in the transmission of sensorystimuli are called primary sensory afferents. The cell bodies for theprimary sensory afferents from the head and some of the internal organsreside in various ganglia associated with the cranial nerves,particularly the trigeminal nuclei and the nucleus of the solitarytract. The cell bodies for the primary sensory afferents for theremainder of the body lie in the dorsal root ganglia of the spinalcolumn. The primary sensory afferents and their processes have beenclassified histologically; the cell bodies fall into two classes: (1)A-types, and (2) B-types. The cell bodies of A-types are relativelylarge (60-120 micrometer in diameter) while the cell bodies of B-typesare smaller (14-30 micrometer) and more numerous. Similarly theprocesses fall into two categories: (1) C-fibers, and (2) A-fibers.C-fibers lack the myelin sheath that A-fibers possess. A-fibers can befurther sub-divided into A beta-fibers, that are large diameters withwell developed myelin, and A delta-fibers, that are thinner with lesswell developed myelin. It is generally believed that A beta-fibers arisefrom A-type cell bodies and that A delta- and C-fibers arise from B-typecell bodies.

[0007] After the activation of the primary sensory afferents the nextstep in the transduction of sensory signals is the activation of theprojection neurons, which carry the signal, via the spinothalamic tract,to higher parts of the central nervous system such as the thalamicnuclei. The cell bodies of these neurons (other than those related tothe cranial nerves) are located in the dorsal horn of the spinal cord.This is also where the synapses between the primary afferents and theprojection neurons are located. The dorsal horn is organized into aseries of laminae that are stacked, with lamina I being most dorsalfollowed by lamina 11, etc. The different classes of primary afferentsmake synapses in different laminae. For cutaneous primary afferents,C-fibers make synapses in laminae I and II, A delta-fibers in laminae I,II, and V, and A beta-fibers in laminae III, IV, and V. Deeper laminae(V-VI I, X) are thought to be involved in the sensory pathways arrivingfrom deeper tissues such as muscles and the viscera.

[0008] The predominant neurotransmitters at the synapses between primaryafferents and projection neurons are substance P, glutamate,calcitonin-gene related peptide (CGRP), and neuropeptide Y. Theefficiency of transmission of these synapses can be altered viadescending pathways and by local interneurons in the spinal cord. Thesemodulatory neurons release a number of mediators that are eitherinhibitory (e.g. opioid peptides, glycine) or excitatory (e.g. nitricoxide, cholecystokinin), to provide a mechanism for enhancing orreducing awareness of sensations.

[0009] Effective pain alleviating drugs are needed. It is known thatintraspinal administration of opioids, such as morphine and fentanyl canalleviate pain. See e.g. Gianno, J., et al., Intrathecal Drug Therapyfor Spasticity and Pain, Springer-Verlag (1996) (which publication isincorporated herein by reference in its entirety). Unfortunately,current drugs used in intraspinal, or intrathecal, injections typicallyhave only short lived antinociceptive effects. As a result, these drugshave to be frequently administered, such as by the aid of a pump forcontinuous infusion. For example, one frequently used pump is theSynchroMed® Infusion System, a programmable, implanted pump availablefrom Medtronic, Inc., of Minneapolis, Minn. However, complications canarise due to the required surgical implantation procedure for the use ofthe pump and the known intrathecally administered drugs for pain, suchas opioids, have the disadvantages of dependency and potentialrespiratory depression.

[0010] Longer acting analgesics are also known, for example, blocks byphenol injection. However, such treatments raise a considerable risk ofirreversible functional impairment.

[0011] Intrathecal administration of botulinum toxin type B to mice totreat thermal hyperalgesia is known. Br. J. Pharmacol1999;127(2):449-456. Additionally, it has been reported (Science, 1999;286:1558-1561) (“Nichols et al.”) that intrathecal injection of acytotoxic saporin-substance P (saporin can be abbreviated as “SAP” andsubstance P can be abbreviated as “SP”) conjugate (which can beabbreviated as SAP-SP) results in a reduction of thermal hyperalgesiaand mechanical allodynia.

[0012] As discussed Nichols et al, supra, spinothalamic andspinoparabrachial neurons are involved in the ascending conduction ofacute noxious stimuli. Apparently, these neurons are projection neuronscan be targeted by substance P. When a conjugate of theribosome-inactivating protein saporin and SP was intrathecally infusedinto the spinal cord, the SAP-SP conjugate is stated to havespecifically concentrated in the projection neurons, apparently becausethese neurons express cell surface receptors for substance P (asubstance P receptor can be abbreviated as “SPR”). Unfortunately, theSAP-SP targeted neurons are killed by the SAP.

[0013] Although SAP-SP is specific for projection neurons becauseprojection neurons appear to express the SPR, an intrathecal injectionof SAP-SP may cause necrosis of other neurons through non-specific orlow affinity SAP-SP neuronal interactions. For example, SAP-SP mayinteract with and cause motor neurons cell death. Since motor neuronsand most other neurons in the spinal cord do not regenerate, it iscontraindicated to use SAP-SP in humans, unless destruction of theneurons with the resulting in permanent disablement, and for example,paralysis, is a desired end result. Clearly it would be desirable to beable to treat pain, including chronic pain, without causing necrosis andirreversible loss of the neurons treated.

[0014] Botulinum Toxin

[0015] The anaerobic, gram positive bacterium Clostridium botulinumproduces a potent polypeptide neurotoxin, botulinum toxin, which causesa neuroparalytic illness in humans and animals referred to as botulism.The spores of Clostridium botulinum are found in soil and can grow inimproperly sterilized and sealed food containers of home basedcanneries, which are the cause of many of the cases of botulism. Theeffects of botulism typically appear 18 to 36 hours after eating thefoodstuffs infected with a Clostridium botulinum culture or spores. Thebotulinum toxin can apparently pass unattenuated through the lining ofthe gut and attack peripheral motor neurons. Symptoms of botulinum toxinintoxication can progress from difficulty walking, swallowing, andspeaking to paralysis of the respiratory muscles and death.

[0016] Botulinum toxin type A is the most lethal natural biologicalagent known to man. About 50 picograms of botulinum toxin (purifiedneurotoxin complex) type A is a LD₅₀ in mice. One unit (U) of botulinumtoxin is defined as the LD₅₀ upon intraperitoneal injection into femaleSwiss Webster mice weighing 18-20 grams each. In other words, one unitof botulinum toxin is the amount of botulinum toxin that kills 50% ofthe group of female Swiss Webster mice. Seven immunologically distinctbotulinum neurotoxins have been characterized, these being respectivelybotulinum neurotoxin serotypes A, B, C₁, D, E, F and G each of which isdistinguished by neutralization with type-specific antibodies. Thedifferent serotypes of botulinum toxin vary in the animal species thatthey affect and in the severity and duration of the paralysis theyevoke. For example, it has been determined that botulinum toxin type Ais 500 times more potent, as measured by the rate of paralysis producedin the rat, than is botulinum toxin type B. Additionally, botulinumtoxin type B has been determined to be non-toxic in primates at a doseof 480 U/kg which is about 12 times the primate LD₅₀ for botulinum toxintype A. Botulinum toxin apparently binds with high affinity tocholinergic motor neurons, is translocated into the neuron and blocksthe release of acetylcholine.

[0017] Botulinum toxins have been used in clinical settings for thetreatment of neuromuscular disorders characterized by hyperactiveskeletal muscles. Botulinum toxin type A has been approved by the U.S.Food and Drug Administration for the treatment of blepharospasm,strabismus, and hemifacial spasm. Non-type A botulinum toxin serotypesapparently have a lower potency and/or a shorter duration of activity ascompared to botulinum toxin type A. Clinical effects of peripheralintramuscular botulinum toxin type A are usually seen within one week ofinjection. The typical duration of symptomatic relief from a singleintramuscular injection of botulinum toxin type A averages about threemonths.

[0018] Although all the botulinum toxins serotypes apparently inhibitrelease of the neurotransmitter acetylcholine at the neuromuscularjunction, they do so by affecting different neurosecretory proteinsand/or cleaving these proteins at different sites. For example,botulinum types A and E both cleave the 25 kiloDalton (kD) synaptosomalassociated protein (SNAP-25), but they target different amino acidsequences within this protein. Botulinum toxin types B, D, F and G acton vesicle-associated protein (VAMP, also called synaptobrevin), witheach serotype cleaving the protein at a different site. Finally,botulinum toxin type C₁ has been shown to cleave both syntaxin andSNAP-25. These differences in mechanism of action may affect therelative potency and/or duration of action of the various botulinumtoxin serotypes.

[0019] Regardless of serotype, the molecular mechanism of toxinintoxication appears to be similar and to involve at least three stepsor stages. In the first step of the process, the toxin binds to thepresynaptic membrane of the target neuron through a specific interactionbetween the H chain and a cell surface receptor; the receptor is thoughtto be different for each type of botulinum toxin and for tetanus toxin.The carboxyl end segment of the H chain, H_(C), appears to be importantfor targeting of the toxin to the cell surface.

[0020] In the second step, the toxin crosses the plasma membrane of thepoisoned cell. The toxin is first engulfed by the cell throughreceptor-mediated endocytosis, and an endosome containing the toxin isformed. The toxin then escapes the endosome into the cytoplasm of thecell. This last step is thought to be mediated by the amino end segmentof the H chain, H_(N), which triggers a conformational change of thetoxin in response to a pH of about 5.5 or lower. Endosomes are known topossess a proton pump which decreases intra endosomal pH. Theconformational shift exposes hydrophobic residues in the toxin, whichpermits the toxin to embed itself in the endosomal membrane. The toxinthen translocates through the endosomal membrane into the cytosol.

[0021] The last step of the mechanism of botulinum toxin activityappears to involve reduction of the disulfide bond joining the H and Lchain. The entire toxic activity of botulinum and tetanus toxins iscontained in the L chain of the holotoxin; the L chain is a zinc (Zn++)endopeptidase which selectively cleaves proteins essential forrecognition and docking of neurotransmitter-containing vesicles with thecytoplasmic surface of the plasma membrane, and fusion of the vesicleswith the plasma membrane. Tetanus neurotoxin, botulinum toxin/B/D,/F,and/G cause degradation of synaptobrevin (also called vesicle-associatedmembrane protein (VAMP)), a synaptosomal membrane protein. Most of theVAMP present at the cytosolic surface of the synaptic vesicle is removedas a result of any one of these cleavage events. Each toxin specificallycleaves a different bond.

[0022] The molecular weight of the botulinum toxin protein molecule, forall seven of the known botulinum toxin serotypes, is about 150 kD.Interestingly, the botulinum toxins are released by Clostridialbacterium as complexes comprising the 150 kD botulinum toxin proteinmolecule along with associated non-toxin proteins. Thus, the botulinumtoxin type A complex can be produced by Clostridial bacterium as 900 kD,500 kD and 300 kD forms. Botulinum toxin types B and C₁ is apparentlyproduced as only a 500 kD complex. Botulinum toxin type D is produced asboth 300 kD and 500 kD complexes. Finally, botulinum toxin types E and Fare produced as only approximately 300 kD complexes. The complexes (i.e.molecular weight greater than about 150 kD) are believed to contain anon-toxin hemaglutinin protein and a non-toxin and non-toxicnonhemaglutinin protein. These two non-toxin proteins (which along withthe botulinum toxin molecule comprise the relevant neurotoxin complex)may act to provide stability against denaturation to the botulinum toxinmolecule and protection against digestive acids when toxin is ingested.Additionally, it is possible that the larger (greater than about 150 kDmolecular weight) botulinum toxin complexes may result in a slower rateof diffusion of the botulinum toxin away from a site of intramuscularinjection of a botulinum toxin complex.

[0023] In vitro studies have indicated that botulinum toxin inhibitspotassium cation induced release of both acetylcholine andnorepinephrine from primary cell cultures of brainstem tissue.Additionally, it has been reported that botulinum toxin inhibits theevoked release of both glycine and glutamate in primary cultures ofspinal cord neurons and that in brain synaptosome preparations botulinumtoxin inhibits the release of each of the neurotransmittersacetylcholine, dopamine, norepinephrine, CGRP and glutamate.

[0024] Botulinum toxin type A can be obtained by establishing andgrowing cultures of Clostridium botulinum in a fermenter and thenharvesting and purifying the fermented mixture in accordance with knownprocedures. All the botulinum toxin serotypes are initially synthesizedas inactive single chain proteins which must be cleaved or nicked byproteases to become neuroactive. The bacterial strains that makebotulinum toxin serotypes A and G possess endogenous proteases andserotypes A and G can therefore be recovered from bacterial cultures inpredominantly their active form. In contrast, botulinum toxin serotypesC₁, D and E are synthesized by nonproteolytic strains and are thereforetypically unactivated when recovered from culture. Serotypes B and F areproduced by both proteolytic and nonproteolytic strains and thereforecan be recovered in either the active or inactive form. However, eventhe proteolytic strains that produce, for example, the botulinum toxintype B serotype only cleave a portion of the toxin produced. The exactproportion of nicked to unnicked molecules depends on the length ofincubation and the temperature of the culture. Therefore, a certainpercentage of any preparation of, for example, the botulinum toxin typeB toxin is likely to be inactive, possibly accounting for the knownsignificantly lower potency of botulinum toxin type B as compared tobotulinum toxin type A. The presence of inactive botulinum toxinmolecules in a clinical preparation will contribute to the overallprotein load of the preparation, which has been linked to increasedantigenicity, without contributing to its clinical efficacy.Additionally, it is known that botulinum toxin type B has, uponintramuscular injection, a shorter duration of activity and is also lesspotent than botulinum toxin type A at the same dose level.

[0025] It has been reported that botulinum toxin type A has been used inclinical settings as follows:

[0026] (1) about 75-125 units of BOTOX®¹ per intramuscular injection(multiple muscles) to treat cervical dystonia;

[0027] (2) 5-10 units of BOTOX® per intramuscular injection to treatglabellar lines (brow furrows) (5 units injected intramuscularly intothe procerus muscle and 10 units injected intramuscularly into eachcorrugator supercilii muscle);

[0028] (3) about 30-80 units of BOTOX® to treat constipation byintrasphincter injection of the puborectalis muscle;

[0029] (4) about 1-5 units per muscle of intramuscularly injected BOTOX®to treat blepharospasm by injecting the lateral pre-tarsal orbicularisoculi muscle of the upper lid and the lateral pre-tarsal orbicularisoculi of the lower lid.

[0030] (5) to treat strabismus, extraocular muscles have been injectedintramuscularly with between about 1-5 units of BOTOX®, the amountinjected varying based upon both the size of the muscle to be injectedand the extent of muscle paralysis desired (i.e. amount of dioptercorrection desired).

[0031] (6) to treat upper limb spasticity following stroke byintramuscular injections of BOTOX® into five different upper limb flexormuscles, as follows:

[0032] (a) flexor digitorum profundus: 7.5 U to 30 U

[0033] (b) flexor digitorum sublimus: 7.5 U to 30 U

[0034] (c) flexor carpi ulnaris: 10 U to 40 U

[0035] (d) flexor carpi radialis: 15 U to 60 U

[0036] (e) biceps brachii: 50 U to 200 U. Each of the five indicatedmuscles has been injected at the same treatment session, so that thepatient receives from 90 U to 360 U of upper limb flexor muscle BOTOX®by intramuscular injection at each treatment session.

[0037] The success of botulinum toxin type A to treat a variety ofclinical conditions has led to interest in other botulinum toxinserotypes. A study of two commercially available botulinum type Apreparations (BOTOX® and Dysport®) and preparations of botulinum toxinstype B and F (both obtained from Wako Chemicals, Japan) has been carriedout to determine local muscle weakening efficacy, safety and antigenicpotential. Botulinum toxin preparations were injected into the head ofthe right gastrocnemius muscle (0.5 to 200.0 units/kg) and muscleweakness was assessed using the mouse digit abduction scoring assay(DAS). ED₅₀ values were calculated from dose response curves. Additionalmice were given intramuscular injections to determine LD₅₀ doses. Thetherapeutic index was calculated as LD₅₀/ED₅₀. Separate groups of micereceived hind limb injections of BOTOX® (5.0 to 10.0 units/kg) orbotulinum toxin type B (50.0 to 400.0 units/kg), and were tested formuscle weakness and increased water consumption, the later being aputative model for dry mouth. Antigenic potential was assessed bymonthly intramuscular injections in rabbits (1.5 or 6.5 ng/kg forbotulinum toxin type B or 0.15 ng/kg for BOTOX®). Peak muscle weaknessand duration were dose related for all serotypes. DAS ED₅₀ values(units/kg) were as follows: BOTOX®: 6.7, Dysport®: 24.7, botulinum toxintype B: 27.0 to 244.0, botulinum toxin type F: 4.3. BOTOX® had a longerduration of action than botulinum toxin type B or botulinum toxin typeF. Therapeutic index values were as follows: BOTOX®: 10.5, Dysport®:6.3, botulinum toxin type B: 3.2. Water consumption was greater in miceinjected with botulinum toxin type B than with BOTOX®, althoughbotulinum toxin type B was less effective at weakening muscles. Afterfour months of injections, 2 of 4 (where treated with 1.5 ng/kg) and 4of 4 (where treated with 6.5 ng/kg) rabbits developed antibodies againstbotulinum toxin type B. In a separate study, 0 of 9 BOTOX® treatedrabbits demonstrated antibodies against botulinum toxin type A. DASresults indicate relative peak potencies of botulinum toxin type A beingequal to botulinum toxin type F, and botulinum toxin type F beinggreater than botulinum toxin type B. With regard to duration of effect,botulinum toxin type A was greater than botulinum toxin type B, andbotulinum toxin type B duration of effect was greater than botulinumtoxin type F. As shown by the therapeutic index values, the twocommercial preparations of botulinum toxin type A (BOTOX® and Dysport®)are different. The increased water consumption behavior observedfollowing hind limb injection of botulinum toxin type B indicates thatclinically significant amounts of this serotype entered the murinesystemic circulation. The results also indicate that in order to achieveefficacy comparable to botulinum toxin type A, it is necessary toincrease doses of the other serotypes examined. Increased dosage cancomprise safety. Furthermore, in rabbits, type B was more antigenic thanwas BOTOX®, possibly because of the higher protein load injected toachieve an effective dose of botulinum toxin type B.

[0038] The tetanus neurotoxin acts mainly in the central nervous system,while botulinum neurotoxin acts at the neuromuscular junction; both actby inhibiting acetylcholine release from the axon of the affected neuroninto the synapse, resulting in paralysis. The effect of intoxication onthe affected neuron is long-lasting and until recently has been thoughtto be irreversible. The tetanus neurotoxin is known to exist in oneimmunologically distinct type

[0039] Acetylcholine

[0040] Typically only a single type of small molecule neurotransmitteris released by each type of neuron in the mammalian nervous system. Theneurotransmitter acetylcholine is secreted by neurons in many areas ofthe brain, but specifically by the large pyramidal cells of the motorcortex, by several different neurons in the basal ganglia, by the motorneurons that innervate the skeletal muscles, by the preganglionicneurons of the autonomic nervous system (both sympathetic andparasympathetic), by the postganglionic neurons of the parasympatheticnervous system, and by some of the postganglionic neurons of thesympathetic nervous system. Essentially, only the postganglionicsympathetic nerve fibers to the sweat glands, the piloerector musclesand a few blood vessels are cholinergic and most of the postganglionicneurons of the sympathetic nervous system secret the neurotransmitternorepinephine. In most instances acetylcholine has an excitatory effect.However, acetylcholine is known to have inhibitory effects at some ofthe peripheral parasympathetic nerve endings, such as inhibition of theheart by the vagal nerve.

[0041] The efferent signals of the autonomic nervous system aretransmitted to the body through either the sympathetic nervous system orthe parasympathetic nervous system. The preganglionic neurons of thesympathetic nervous system extend from preganglionic sympathetic neuroncell bodies located in the intermediolateral horn of the spinal cord.The preganglionic sympathetic nerve fibers, extending from the cellbody, synapse with postganglionic neurons located in either aparavertebral sympathetic ganglion or in a prevertebral ganglion. Since,the preganglionic neurons of both the sympathetic and parasympatheticnervous system are cholinergic, application of acetylcholine to theganglia will excite both sympathetic and parasympathetic postganglionicneurons.

[0042] Acetylcholine activates two types of receptors, muscarinic andnicotinic receptors. The muscarinic receptors are found in all effectorcells stimulated by the postganglionic neurons of the parasympatheticnervous system, as well as in those stimulated by the postganglioniccholinergic neurons of the sympathetic nervous system. The nicotinicreceptors are found in the synapses between the preganglionic andpostganglionic neurons of both the sympathetic and parasympathetic. Thenicotinic receptors are also present in many membranes of skeletalmuscle fibers at the neuromuscular junction.

[0043] Acetylcholine is released from cholinergic neurons when small,clear, intracellular vesicles fuse with the presynaptic neuronal cellmembrane. A wide variety of non-neuronal secretory cells, such as,adrenal medulla (as well as the PC12 cell line) and pancreatic isletcells release catecholamines and insulin, respectively, from largedense-core vesicles. The PC12 cell line is a clone of ratpheochromocytoma cells extensively used as a tissue culture model forstudies of sympathoadrenal development. Botulinum toxin inhibits therelease of both types of compounds from both types of cells in vitro,permeabilized (as by electroporation) or by direct injection of thetoxin into the denervated cell. Botulinum toxin is also known to blockrelease of the neurotransmitter glutamate from cortical synaptosomescell cultures.

[0044] U.S. Pat. No. 5,989,545 (“Foster et al.”) (incorporated herein byreference in its entirety) discusses conjugating clostridial neurotoxinsto targeting moieties in order to direct the inhibitory effect ofclostridial neurotoxins toward primary sensory afferent neurons. Thus,the mechanism by which the agents disclosed by Foster et al alleviatepain is as follows: the targeting moieties of the agents, for examplethe growth factors, bind to receptor sites on the sensory afferent nerveterminals, for example the growth factor receptors, in the spinal cord;then, the clostridial neurotoxins, along with the conjugated targetingmoieties, translocate into the nerve terminal and inhibit the release ofone or more transmitters involved in the signaling of pain, and therebyalleviate pain.

[0045] Unlike SAP-SP, the clostridial-targeting moiety conjugatesdisclosed by Foster et al do not appear to be cytotoxic. Despite theirsuperiority to the SAP-SP in that they are non-cytotoxic, they are stillinadequate as pain alleviating agents because they lack the specificityfor treating pain. More particularly, the Foster et al's targetingmoieties intended for primary sensory afferent neurons are non-specific.

[0046] Thus, the agents disclosed by Foster et al are non-specificbecause their targeting moieties are not known to bind to receptorsspecifically and to primarily localize to primary sensory afferent nerveterminals. Therefore, the targeting moieties disclosed by Foster et al.may readily bind to receptors on neuronal terminals, or neurons, thatare not primary sensory afferent synaptic terminals. For example, thetargeting moiety comprising nerve growth factor disclosed by Foster mayreadily bind to receptors on nerve terminals and neurons other than thereceptors on the primary sensory afferent nerve terminals, because nervegrowth factor receptors are found on most neurons. As such, theclostridial neurotoxin conjugate disclosed by Foster et al may bind toone of these other neurons, for example the neurons involved in thesympathetic pathway, translocate into their cytosol, inhibit the releaseof their neurotransmitters, and thereby inhibiting their functions. Suchrandom, non-specific inhibition may cause undesirable side effectsduring the treatment of pain.

[0047] Similarly, bradykinin, another targeting moieties disclosed byFoster et al, have been shown to have high density concentration in themotor neurons of the ventral horn in the spinal cord. (See Lopes et al,Neuroscience 78(2):481-497, the content of which is incorporated in itsentirety herein by reference.) Agents disclosed by Foster et al whichbear bradykinins as targeting moieties will significantly interact andinterfere with motor functions when the agents are injectedintraspinally to treat pain.

[0048] Also, the opioid receptor binding targeting moieties disclosed byFoster et al, for example, methionine-enkephalin, are non-specific withrespect to directing the clostridial neurotoxin to the primary sensoryafferent nerve terminal. Kandel et al, Principles of Neural Science,third edition, page 395,(1991), indicated that opioid receptors arewidely distributed throughout the central nervous system, suggestingthat opioid receptors, when activated, modulate physiological functionsother than pain. Therefore, the clostridial neurotoxin-targeting moiety,as disclosed by Foster el al, may bind to and interfere with cellshaving opioid receptors but are not involved in the pain pathway. Whenthis non-specific binding and interference occur, undesirous sideeffects may result.

[0049] What is needed therefore is an specific (high affinity)therapeutically effective, long duration non-cytotoxic agent and methodfor treating pain.

SUMMARY

[0050] The present invention meets this need by providing specific (highaffinity) therapeutically effective, long duration non-cytotoxic agentsand methods for treating pain. I have discovered agents effective inalleviating pain, methods of making such agents and methods of usingsuch agents to alleviate pain. The present invention providesnon-cytotoxic agents for treating pain which preferably have one or moreof the characteristics of long duration of activity and specificity forthe treatment of pain with limited or substantially insignificant sideeffects at therapeutic dose levels. Furthermore, the methods ofproducing these agents are relatively straight forward and effective toprovide the desired results.

[0051] In one embodiment of the invention, methods for treating pain ina subject, comprise administering an agent to the subject, wherein theagent comprises a botulinum toxin component covalently coupled tosubstance P. In a further embodiment, the botulinum toxin componentcomprises the proteolytic domain of the botulinum toxin. In anotherembodiment, the botulinum toxin component comprises the proteolyticdomain and translocational domain of the botulinum toxin. In yet afurther embodiment, the proteolytic domain is covalently coupled to thetranslocational domain.

[0052] In another embodiment, the invention provides methods forreducing pain in a subject, comprising administering an agent to asubject, wherein the agent comprises a botulinum toxin component coupledto substance P. In yet another embodiment, the agent comprises abotulinum toxin proteolytic domain covalently attached to a botulinumtoxin translocational domain, and substance P covalently attached to thetranslocational domain.

[0053] In practicing the foregoing methods, the botulinum toxincomponent is preferably selected from a group of botulinum toxinserotypes consisting of serotype A, serotype B, serotype C₁, serotype D,serotype E, serotype F, and serotype G. In a preferred embodiment, thebotulinum toxin component is botulinum toxin serotype A. In oneembodiment of the invention, the botulinum toxin component comprises anH_(N) and an L chain of the botulinum toxin. The H_(N) chain ispreferably obtained from a botulinum toxin selected from the groupconsisting of botulinum toxin serotype A, serotype B, serotype C₁,serotype D, serotype E, serotype F, and serotype G.

[0054] In yet another embodiment of the invention, methods for reducingpain in a subject, comprise administering an agent to the subject,wherein the agent comprises a botulinum toxin type A proteolytic domaincovalently attached to a botulinum toxin type A translocational domain,and substance P covalently attached to the translocational domain.

[0055] In additional embodiments of the invention, the botulinum toxincomponent may be coupled to a precursor or analogues of substance P.

[0056] In a preferred embodiment of the invention, the proteolyticdomain is a botulinum toxin type A proteolytic domain. In yet anotherembodiment of the invention, the translocational domain is a botulinumtoxin type A translocational domain. In a further embodiment of theinvention, the proteolytic and translocational domains are botulinumtoxin type A proteolytic and translocational domains. In practicing theforegoing methods, the agent may be administered before, or after, theonset of a nociceptive event experienced by the subject. An example of anociceptive event includes a neuropathic pain syndrome, such asinflammatory pain.

[0057] In practicing the foregoing methods, the agent of the inventioncan be administered intramusclarly and/or intrathecally. The amount ofagent administered may reduce the pain in a subject by about 20%. In apreferred embodiment, the agent reduces the pain in the patient by about50%. In a more preferred embodiment, the agent reduces the pain in thepatient by about 80%.

[0058] In another broad aspect of this invention, there are providedmethods for treatment of a bone tumor. A preferable treatment of bonetumor includes the treatment of, for example, pain associated with thebone tumor, 1s which comprise administering effective doses of theagents according to the invention. The routes of administrationpreferably include administration locally to the peripheral location ofpain, particularly to a bone tumor or the vicinity of a bone tumor.

[0059] Each and every feature described herein, and each and everycombination of two or more of such features, is included within thescope of the present invention provided that the features included insuch a combination are not mutually inconsistent.

[0060] Definitions

[0061] Light chain means the light chain of a clostridial neurotoxin. Ithas a molecular weight of about 50 kDa, and can be referred to as Lchain, L, or as the proteolytic domain (amino acid sequence) of aclostridial neurotoxin.

[0062] Heavy chain means the heavy chain of a clostridial neurotoxin. Ithas a molecular weight of about 100 kDa and can be referred to as Hchain, or as H.

[0063] H_(C) means a fragment (about 50 kDa) derived from the H chain ofa clostridial neurotoxin which is approximately equivalent to thecarboxyl end segment of the H chain, or the portion corresponding tothat fragment in the intact H chain. It is believed to be immunogenicand to contain the portion of the natural or wild type clostridialneurotoxin involved in high affinity, presynaptic binding to motorneurons.

[0064] H_(N) means a fragment (about 50 kDa) derived from the H chain ofa clostridial neurotoxin which is approximately equivalent to the aminoend segment of the H chain, or the portion corresponding to thatfragment in the intact in the H chain. It is believed to contain theportion of the natural or wild type clostridial neurotoxin involved inthe translocation of the L chain across an intracellular endosomalmembrane.

[0065] LH_(N) or L-H_(N) means a fragment derived from a clostridialneurotoxin that contains the L chain, or a functional fragment thereof,coupled to the H_(N) domain. It can be obtained from the intactclostridial neurotoxin by proteolysis, so as to remove or to modify theH_(C) domain.

[0066] Targeting moiety means a molecule that has a specific bindingaffinity for a cell surface receptor, for example, for a neuronalreceptor so as to influence the transmission or reception of painsignals by the neuron. “About” means approximately or nearly and in thecontext of a numerical value set forth herein means ±10% of thenumerical value or range recited or claimed.

[0067] “Bone tumor” means a neoplasm located on or within a bone.

[0068] “Local administration” means direct administration by anon-systemic route at or in the vicinity of the site of an affliction,disorder, or perceived pain.

[0069] As used herein, an “agent” is defined as a modified neurotoxinthat possesses biological activity that is similar, or substantiallysimilar, to a biological activity of the unmodified neurotoxin. Themodified neurotoxin is said to be “substantially similar” to anotherneurotoxin if both neurotoxins have similar structures, or if bothneurotoxins possess a similar biological activity. For example, abiological activity of a neurotoxin includes the binding of theneurotoxin to a receptor on a neural cell, and interfering with thenormal function of the neural cell. Examples of normal functions ofneurons include action potential discharge, and neurotransmission,including synaptic transmission. A neurotoxin can inhibit actionpotential discharge. A neurotoxin may also inhibit synapticneurotransmission. One example of a neurotoxin is a botulinum toxin. Amodified neurotoxin is a neurotoxin that has been changed from itsnatural state. Modified neurotoxins include analogs and fragments ofneurotoxins. Modified neurotoxins can be naturally-made orrecombinantly-made. One example of a modified neurotoxin, as disclosedherein, is botulinum toxin coupled to a substance P molecule.

[0070] Importantly, the agents disclosed herein are preferablyadministered by local administration, that is directly to the site wherea therapeutic effect is desired.

DESCRIPTION

[0071] This invention is based upon the discovery that pain, includingbone tumor pain, can be treated by administration to a patient of anagent which is comprised of a derivative of a clostridial neurotoxin anda targeting moiety, where the targeting moiety is selected from thegroup consisting of transmission compounds which can be released from aneuron upon the initiation, transmission of, or facilitation of thegeneration of, a pain signal by the neuron.

[0072] Significantly, the agents of the present invention can alleviatepain without being cytotoxic to their target neurons. Furthermore,agents within the scope of the present invention can be administered toboth central nociceptive neurons and to primary sensory afferentneurons.

[0073] The mechanism of action for these agents in alleviating pain iscurrently not fully understood. Without wishing to limit the inventionto any particular theory or mechanism of operation, it is believed that,at least with respect to areas in spinal cord, the agents disclosedherein target neurons having receptors for neurotransmitters that arereleased by neurons for or upon the transmission of pain signals. Forexample, when the targeting moiety is substance P, the agent is thoughtto interact with neurons expressing substance P receptors (SPR), such asprojection neurons. Moreover, the receptors binding neurotransmittersreleased for the transmission of pain are primarily expressed on cellsinvolved in the transmission of pain signals. For example, with respectto the central nervous system, it is well known that substance Preceptors are primarily expressed on projection neurons in the dorsalhorn of the spinal cord. See e.g. Vigna et al, J. Neuroscience,14(2):834-845 (1994).

[0074] Therefore, the agents as described in this invention preferablyare very specific for treating pain because they do not substantially orsignificantly interact and/or interfere with neurons and cells of othersystems. Moreover, it is believed that the agents of this invention mayenter into these specific neurons, for example projection neurons,through an endocytosis process. Once inside the neurons, it is furtherbelieved that the H_(N) of these agents facilitate the translocation ofthe agent into the cytosol. In the cytosol, the agent, or a componentthereof, can inhibit the release of a neurotransmitter involved in thefurther transmission of pain signals. It is further believed that the Lchain of the clostridial neurotoxin component of the agent isresponsible for the inhibition of the release of neurotransmitters thatare involved in pain transmission by interfering with their vesicularexocytosis.

[0075] Additionally, the agents of this invention also provide painalleviating effects when locally applied to peripheral pain sites. Alsowithout wishing to limit the invention to any particular theory ormechanism of operation, it is believed that the agents interfere withthe functions of cells having receptors for nociception, for exampleorphanin, substance P and/or kyotorphin, at the peripheral locations.See Ueda, Jpn J. Pharmacology, 79(3):263-268, the content of which isincorporated in its entirety herein by reference. These cells areuniquely involved in pain transmissions and the disruption of theirfunctions by the agents can result in pain alleviation. Furthermore, itis believed that the mechanism for the inhibitory effects by agents inthese cells is similar to that described above. Moreover, these agentscan also bind, enter into and interfere with the function of primarysensory neurons.

[0076] According to one broad aspect of the invention, the clostridialneurotoxin component is covalently coupled to a targeting moiety. Theclostridial neurotoxin component is a polypeptide and may be derivedfrom Clostridial beratti, Clostridial butyricum, or Clostridialbotulinum. More preferably, the clostridium neurotoxin component isderived from Clostridial botulinum. Clostridial botulinum producesbotulinum toxin types A, B, C, D, E, F and G. Although any of thesetoxin types may be used in the present invention, botulinum type A ismore preferably used.

[0077] Furthermore, the clostridial neurotoxin component may compriseonly a fragment of the entire neurotoxin. For example, it is known inthe art that the H_(C) of the neurotoxin molecule can be removed fromthe other segment of the H chain, the H_(N), such that the H_(N)fragment remains disulphide linked to the L chain of the neurotoxinmolecule to provide a fragment known as the LH_(N). Thus, in oneembodiment of the present invention the LH_(N) fragment of a clostridialneurotoxin is covalently coupled, using linkages which may include oneor more spacer regions, to a targeting moiety.

[0078] In another embodiment of the invention, the domain having theH_(C) of a clostridial neurotoxin is removed, mutated or modified, e.g.by chemical modification, to reduce, or preferably incapacitate, itsability to bind the neurotoxin to receptors at the neuromuscularjunction. This modified clostridial neurotoxin is then covalentlycoupled, using linkages which may include one or more spacer regions, toa targeting moiety.

[0079] In another embodiment of the invention, the H chain of aclostridial neurotoxin, in which the H_(C) is removed, mutated ormodified, e.g. by chemical modification, to reduce, preferablyincapacitate, its ability to bind the neurotoxin to receptors at theneuromuscular junction is combined with the L-chain of a differentclostridial neurotoxin, to form a hybrid. For example, in oneembodiment, the clostridial neurotoxin component comprises an H chainwith the H_(C) removed, mutated or modified derived from botulinum toxintype A, and an L chain derived from another botulinum toxin type. Thedescribed hybrid is covalently coupled to a targeting moiety, preferablywith one or more spacer regions.

[0080] In another embodiment of the invention the L chain of aclostridial neurotoxin, or a fragment of the L chain containing theendopeptidase activity, is linked, using linkages which may include oneor more spacer regions, to a targeting moiety which can also effect theinternalization of the L chain, or fragment thereof containingendopeptidase activity, into the cytoplasm of the cell.

[0081] In a preferred embodiment, the agent comprises the H_(N) the Lchain and the targeting moiety, covalently linked together. Thetargeting moiety according to the first aspect of the invention ispreferably derived from amino acids, substituted counterparts thereofand mixtures thereof. The term “substituted counterparts thereof” as itrelates to any of the above noted amino acids refers to molecules thatare functionally and physically similar to the amino acids, either asindependent units or units incorporated into macromolecules, forexample, peptides.

[0082] In one preferred aspect of the present invention, the targetingmoiety is glutamate, since glutamate is the predominant neurotransmitterat the synapses between primary afferents and projection neurons. Inanother embodiment, the targeting moieties may be components that aresubstantially similar to the transmission compounds, for example,glutamate, in this particular instance. Hereinafter, the term“components that are substantially similar to the transmissioncompounds,” is defined as molecules or substances that have the samefunctions as that of the transmission compounds, for example, binding toreceptors that are involved in the transmission of pain signals.

[0083] In one embodiment, components that are substantially similar toglutamate are agonists of glutamate. For example, componentssubstantially similar to glutamate are quisqualate,DL-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate,N-Me-D-aspartate, kinate and the like. Additionally, componentssubstantially similar to glutamate may also include antagonists ofglutamate. For example, these molecules include6-cyano-7nitroquinozaline-2,3-dione,3-(2-carboxypiperazin-4-yl)propyl-1-phosponic acid, lactonized kainateand the like.

[0084] In a more preferred embodiment, the amino acids link to form oneof the peptides which are released by neurons for the transmission ofpain signals. For example, these peptides include neuropeptide Y andcalcitonin-gene related peptide (CGRP). Even more preferably, thepeptide is substance P.

[0085] In another embodiment, components substantially similar tosubstance P may be used as targeting moieties. These components includesubstance P precursors, fragments, analogs and/or derivatives. Thehistory, isolation, identification, and synthesis of substance P and itsprecursors, fragments, analogs and/or derivatives are disclosed in U.S.Pat. No. 5,891,842 (incorporated herein by reference in its entirety).

[0086] Substance P is an 11 amino acid peptide which has a number ofdifferent natural and synthetic precursor forms; has been demonstratedto be converted into a variety of naturally occurring amino-terminalpeptide fragments; and can be obtained in analog format compromising,substituted counterparts thereof, for example, lysine methyl ester,D-amino acids or disulfide bridges substitutions, thereby yielding morestable and discriminating formulations. A representative listing ofsubstance P and its related chemical entities is provided by Table Ibelow. The amino acid sequence (1) in Table I(Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-amide) can be referred to aSEQ ID NO:1, and the subsequent 17 amino acid sequences set forth inTable one can be similarly identified as SEQ ID NO:2 to SEQ ID NO:18.TABLE 1 Substance P, and Representative Precursors, Fragments andStabilized Or Substituted Analogs Name Formula (1) Substance PArg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly- Leu-Met-amide Natural Precursors:(2) Substance P-Glycine* Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-Gly (3) Substance P- Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Glycine-Lysine* Leu-Met-Gly-Lys (4) Substance P-Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly- Glycine-Lysine Leu-Met-Gly-Lys-ArgArginine* Carboxy-Ester Synthetic Precursors: (5) Substance P-Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly- Glycine Leu-Met-Gly-OMe MethylEster° (6) Substance P- Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Glycine-Lysine Leu-Met-Gly-Lys-OMe Methyl Ester° (7) Substance P-Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly- Glycine-LysineLeu-Met-Gly-Lys-Arg-OMe Arginine Methyl Ester° (8) Substance P-Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe- Glycine Gly-Leu-Met-Gly-OEth EthylEster° (9) Substance P- Arg-Pro-Lys-Pro-Gln-Gln-Phe-.Phe-Gly-Glycine-Lysine Leu-Met-Gly-Lys-OEth Ethyl Ester° (10) Substance P-Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly- Glycine-LysineLeu-Met-Gly-Lys-Arg-OEth Arginine Ethyl Ester° Naturally-OccurringAmino-Terminal Peptide Fragments: (11) Substance P/1-4# Arg-Pro-Lys-Pro(12) Substance P/1-7# Arg-Pro-Lys-Pro-Gln-Gln-Phe (13) Substance P/1-9#Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly Analogs Comprising Synthetic D-AminoAcids Or Disulfide (Cys-Cys) Bridges: (14) [D-Pro2, D-Phe7,Arg-D-Pro-Lys-Pro-Gln-Gln-D-Phe-Phe- D- D-Trp9]- Trp-Leu-Met-amideSubstance P^(¢) (15) [D-Pro2, D-Phe7,Arg-D-Pro-Lys-Pro-Gln-Gln-D-Phe-Phe- D- D-Trp9]- Trp-Leu-Met-Gly(Substance P-Glycine^(¢)) (16) [D-Pro2, D-Trp7,Arg-D-Pro-Lys-Pro-Gln-Gln-D-Trp-Phe- D- D-Trp9]- Trp-Leu-Met-amideSubstance P^(¢) (17) [D-Pro2,D-Trp7,Arg-D-Pro-Lys-Pro-Gln-Gln-D-Trp-Phe- D- D-Trp9]- Trp-Leu-Met-GlySubstance P-Glycine^(¢) (18) [Cys3, Cys6, Tyr8, Pro10]-Arg-Pro-Cys-Pro-Gln-Cys-Phe-Tyr-Gly- Pro Substance P^(¢) Met-amide

[0087] The components substantially similar to substance P may alsoinclude molecules in the same family as that of substance P. Forexample, a preferred family of such molecules would be the tachykininfamily to which substance P is a member. Examples of some family membersof tachykinins include physalaemin, kassinin, uperolein, eledoisin,substance K and the like.

[0088] In a preferred embodiment, the agent comprises a clostridialneurotoxin component, for example LH_(N), coupled to substance P. Inanother preferred embodiment, the agent comprises a hybrid of twoclostridial neurotoxins, such as the H chain, preferably H_(N), derivedfrom botulinum toxin A and the L chain derived from another botulinumtoxin, coupled to substance P. In another preferred embodiment, theclostridial component of the agent is a botulinum toxin type A in whichthe H_(C) has been removed or modified, coupled to substance P.

[0089] In another preferred embodiment, the agent comprises an L chainof a clostridial neurotoxin, or a fragment of the L chain containing theendopeptidase activity, coupled substance P. Even more preferably, the Lchain or fragment of the L chain is derived from botulinum toxin A, andis coupled to substance P. Additionally, it is preferred that the Lchain coupled to the substance P is covalently linked to H_(N).

[0090] The clostridial components and the targeting moieties are coupledby covalent linkages. In a preferred embodiment, the linkages mayinclude appropriate spacer regions. Spacer regions have many functionswithin this invention. For example, one of the functions of the spacerregions is to provide for adequate distance between the clostridialneurotoxin components and the targeting moieties so that the twocomponents can independently and freely move about, without an internalsteric hindrance.

[0091] In one embodiment, the spacer region is made up of sugarmolecules, for example, saccharides, glucose, etc. In anotherembodiment, the spacer region may be constructed from an aliphaticchain. In another embodiment, the spacer regions may be constructed bylinking together a series of amino acids, preferably glycine becausethey are small and are devoid of any functional group. In yet anotherembodiment, the spacer region may comprise one or more of the sugarmolecules, aliphatic chains, and amino acids.

[0092] Also, these agents can be thought of as being polypeptides, witha first and a second amino acid sequence region. The first regionpreferably includes a first domain and a second domain. Preferably, thefirst domain of the first amino acid sequence comprises a targetingmoiety. In one embodiment, the targeting moiety is able to bind tosurface receptors of the spinal cord neurons under physiologicalconditions. More preferably, the targeting moiety specifically binds areceptor on a spinal cord dorsal horn neuron, for example a projectionneuron.

[0093] Preferably, the second domain comprises a heavy chain or aportion thereof of a clostridial neurotoxin. Even more preferably, theH_(N) of the heavy chain is able to facilitate the transfer of thepolypeptide across an endosome membrane into the cytosol of the neuron.In one embodiment, the second domain of the first amino acid sequencecomprises a clostridial neurotoxin heavy chain. More preferably, theclostridial neurotoxin heavy chain is derived from Clostridium botulinumneurotoxin type A. Even more preferably, the heavy chain is derived fromthe H_(N) of Clostridium botulinum neurotoxin type A. In yet anotherembodiment, the heavy chain may be derived from Clostridial botulinumtypes B, C, D, E, F, G and mixtures thereof. Also, the heavy chain maybe derived from Clostridial baratii and Clostridial butyricum.Additionally, the heavy chain, preferably the H_(N), may be derived fromClostridial tetani.

[0094] The second amino acid sequence region preferably comprises the Lchain. The L chain is the effective therapeutic element havingbiological activity because, as discussed above, once it is transferredinside the neuron it interferes with the exocytosis process ofneurotransmitter. Preferably, the light chain is derived fromClostridial botulinum neurotoxin type A. According to another broadaspect of this invention recombinant techniques are used to produce theclostridial neurotoxin components of the agents. The technique includessteps of obtaining genetic materials from either DNA cloned from naturalsources, or synthetic oligonucleotide sequences, which have codes forclostridial neurotoxin components including clostridial neurotoxins,modified clostridial neurotoxins and fragments thereof. The geneticconstructs are incorporated into host cells for amplification by firstfusing the genetic constructs with a cloning vectors, such as phages orplasmids. Then the cloning vectors are inserted into hosts, preferablyE. coli's. Following the expressions of the recombinant genes in hostcells, the resultant proteins can be isolated using conventionaltechniques. The clostridial neurotoxin components derived from therecombinant techniques can then be chemically coupled to targetingmoieties. Preferably, the linkages between the clostridial componentsand the targeting moieties include an appropriate spacer regions.

[0095] In another embodiment, the genetic constructs include genescoding for both the clostridial neurotoxin components and the targetingmoieties, for example, forming fusion proteins. Additionally, thegenetic constructs may include genes coding for appropriate spacerregions between the clostridial neurotoxin components and the targetingmoieties. From this aspect, the agents may be thought of as polypeptidescomprising a first amino acid sequence region and a second amino acidsequence region. The first region may further comprise a first domainand a second domain. The details of these regions and domains aredescribed above.

[0096] In another embodiment, the required L-H_(N), which may be ahybrid of an L chain and an H_(N) from different clostridial toxintypes, is expressed recombinantly as a fusion protein. Such LH_(N)hybrid may also be coupled to the targeting moiety, which may furtherinclude one or more spacer regions between them.

[0097] In another embodiment of the invention the L chain of aclostridial neurotoxin, or a fragment of the L chain containing theendopeptidase activity, is expressed recombinantly as a fusion proteinwith the H_(N) of the H chain and the targeting moiety which can alsoaffect the internalization of the L chain, or fragment thereofcontaining the endopeptidase activity, into the cytoplasm of the cell.The expressed fusion protein may also include one or more spacerregions.

[0098] There are many advantages to producing these agentsrecombinantly. For example, production of neurotoxin from anaerobicClostridium cultures is a cumbersome and time-consuming processincluding a multi-step purification protocol involving several proteinprecipitation steps and either prolonged and repeated crystallization ofthe toxin or several stages of column chromatography. Significantly, thehigh toxicity of the product dictates that the procedure must beperformed under strict containment (BL-3). During the fermentationprocess, the folded single-chain neurotoxins are activated by endogenousclostridial proteases through a process termed nicking. This involvesthe removal of approximately 10 amino acid residues from thesingle-chain to create the dichain form in which the two chains remaincovalently linked through the intrachain disulfide bond.

[0099] The nicked neurotoxin is much more active than the unnicked form.The amount and precise location of nicking varies with the serotypes ofthe bacteria producing the toxin. The differences in single-chainneurotoxin activation and, hence, the yield of nicked toxin, are due tovariations in the type and amounts of proteolytic activity produced by agiven strain. For example, greater than 99% of Clostridial botulinumtype A single-chain neurotoxin is activated by the Hall A Clostridialbotulinum strain, whereas type B and E strains produce toxins with loweramounts of activation (0 to 75% depending upon the fermentation time).Thus, the high toxicity of the mature neurotoxin plays a major part inthe commercial manufacture of neurotoxins as therapeutic agents.

[0100] The degree of activation of engineered clostridial toxins is,therefore, an important consideration for manufacture of thesematerials. It would be a major advantage if neurotoxins such asbotulinum toxin and tetanus toxin could be expressed, recombinantly, inhigh yield in rapidly-growing bacteria (such as heterologous E. colicells) as relatively non-toxic single-chains (or single chains havingreduced toxic activity) which are safe, easy to isolate and simple toconvert to the fully-active form.

[0101] With safety being a prime concern, previous work has concentratedon the expression in E. coli and purification of individual H and Lchains of tetanus and botulinum toxins; these isolated chains are, bythemselves, non-toxic; see Li et al., Biochemistry 33:7014-7020 (1994);Zhou et al., Biochemistry 34:15175-15181 (1995), hereby incorporated byreference herein. Following the separate production of these peptidechains and under strictly controlled conditions the H and L subunits canbe combined by oxidative disulphide linkage to form the neuroparalyticdi-chains.

[0102] In another broad aspect of this invention, methods are providedfor the treatment of pain which comprise administering effective dosesof the agents according to the invention. The agents described in thisinvention can be used in vivo, either directly formulated or as apharmaceutically acceptable salt, for treatment of pain.

[0103] For example, in a preferable embodiment, agents according to theinvention can be administered by spinal injection (epidural orintrathecal) at the level of the spinal segment involved in theinnervation of an affected organ for the treatment of pain. This is, forexample, applicable in the treatment of deep tissue pain, such aschronic malignant pain.

[0104] As used herein “intraspinal” means into or within the epiduralspace, the intrathecal space, the white or gray matter of the spinalcord or affiliated structures such as the dorsal root and dorsal rootganglia.

[0105] In another aspect of the invention, there are provided methodsfor treatment of pain which comprise locally administering directly to apainful, benign bone tumor of a human patient therapeutically effectivedoses of an agent in accordance with the invention.

[0106] Examples of neoplasms which can be treated according to thepresent invention are benign bone tumors of cartilaginous origin such asenchondroma, osteochondroma, chondroblastoma and chondromyxoid, all ofcartilaginous origin, as well as benign bone tumors of bone origininclude osteoid osteoma and osteoblastoma. An agent, such as botulinumtoxin-substance P can require according to the methods of the presentinvention, from about 1 to 7 days to achieve an antinociceptive effect,or to begin to achieve a necrotic effect upon a bone tumor. Thus,malignant bone tumors are excluded from the scope of the presentinvention because such tumors are preferably treated by a protocol withimmediate effects such as surgical excision or radiotherapy, so as toprevent the tumor from metastasizing.

[0107] Additionally, an agent according to the present invention may belocally administered in vivo directly to the site of the tumor, whetheron or within a bone. Known local drug administration methods suitablefor this purpose include by long needle for bolus injection and byinsertion of a controlled release implant.

[0108] In one embodiment of the invention, the agent of the inventionreduces pain in the subject. For example, pain is reduced by at least20%. In a more preferred embodiment, the pain in the subject is reducedby at least 50%. In an even more preferred embodiment, the pain isreduced by at least 80%.

[0109] Preferably, clostridial neurotoxin components of agents used topractice a method within the scope of the present invention comprisebotulinum toxins, such as one of the type A, B, C, D, E, F or G.Preferably, the botulinum toxin used is botulinum toxin type A, becauseof its high potency in humans and ready availability. The targetingmoiety of the agents used to practice the method herein is preferably asubstance P.

[0110] An intraspinal route for administration of a neurotoxin accordingto the present disclosed invention can be selected based upon criteriasuch as the solubility characteristics of the agents chosen as well asthe amount of the agents to be administered. The amount of the agentsadministered can vary widely according to the particular disorder beingtreated, its severity and other various patient variables includingsize, weight, age, and responsiveness to therapy. For example, theextent of the area of CNS afferent pain neuron somata influenced isbelieved to be proportional to the volume of agents injected, while thequantity of the analgesia is, for most dose ranges, believed to beproportional to the concentration of agents injected. Furthermore, theparticular intraspinal location for agents administration can dependupon the dermosome location of the pain to be treated. Methods fordetermining the appropriate route of administration and dosage aregenerally determined on a case by case basis by the attending physician.Such determinations are routine to one of ordinary skill in the art (seefor example, Harrison's Principles of Internal Medicine (1998), editedby Anthony Fauci et al., 14^(th) edition, published by McGraw Hill).

[0111] Preferably, the intraspinal administration is carried outintrathecally because of the greater ease in which the relatively largerintrathecal space is accessed and because the preferred agents generallyexhibits low solubility in the lipid rich epidural environment. It isfound that both inflammatory and neuropathic pain can be effectivelytreated by the disclosed methods without significant muscle spasticityor flaccidity or other side effects.

[0112] Intraspinal administration of the agents according to the presentinvention can be by various routes such as by catheterization or byspinal tap injection. The long lasting nature of the therapeutic effectsof the present invention substantially removes the need for chronicantinociceptive drug administration, so that the present methods areadvantageously practiced by infrequent spinal tap injection of theagents. Additionally, an intrathecal spinal tap agents administrationroute facilitates a more precise and localized delivery of agents withless danger of damage to the CNS, as compared to moving a catheter toaccess other CNS locations.

[0113] Intrathecal agents can be administered by bolus injection or bycatheterization. The catheter can be inserted at L3-4 or at L4-5, a safedistance from the spinal cord which in humans terminates at L1, andguided upward in the subarachnoid space to rest at the desired site. Forpain management, placement of the catheter or location of bolusinjection by syringe depends on the site of the perceived pain, and thephysicians preference. It is important to note that therapeutic agentadministration according to the present disclosed methods can be carriedout before the occurrence of, or during the experience of a nociceptiveevent or syndrome.

[0114] It is found that an agent, such as the LH_(N) (derived frombotulinum toxin type A)-substance P, can be intraspinally administeredaccording to the present disclosed methods in amounts between about 1 Uto about 500 U. Preferably the amounts are between about 10 U and about300 U. More preferably the amount is between about 10 and about 200 U,such as about 70 U.

[0115] In a human patient, the therapeutically effective doses (when theclostridial neurotoxin component is derived from a botulinum toxin typeA) can be amounts between about 10⁻³ U/kg and about 35 U/kg. A dose ofabout 10⁻³ U/kg can result in an antinociceptive effect if delivereddirectly to or onto the dorsal horn of the CNS and/or if agents deliveryis assisted by methods such as iontophoresis. Intraspinal administrationof less than about 10⁻³ U/kg does not result in a significant or lastingtherapeutic result. An intraspinal dose of more than 35 U/kg approachesa lethal dose of an agent such as the L-H_(N) (derived from botulinumtoxin type A)-substance P. It is desired that the agents used to obtaineither antinociceptive effect contact the nerves of the CNS so as tofavorably influence or down regulate the perception of pain in theinnervated organ or tissue. Thus, intraspinal administration of agentsby, for example, epidural injection can require an increase of thedosage by a factor of about ten to account for dilution of the agentsupon diffusion from the epidural space to the intrathecal space andthence to the exterior nerves of the CNS.

[0116] A preferred range for intrathecal administration of an agent,such as the LH_(N)(type A)-substance P, so as to achieve anantinociceptive effect in the patient treated is from about 10⁻² U/kg toabout 10 U/kg. A more preferred range for intrathecal administration ofan agent, such as the LH_(N) (derived from botulinum toxin typeA)-substance P, so as to achieve an antinociceptive effect in thepatient treated is from about 10⁻U/kg to about 10 U/kg. Less than about10⁻¹ U/kg can result in the desired therapeutic effect being of lessthan the optimal or longest possible duration, while more than about 10U/kg can still result in some symptoms of muscle flaccidity. A mostpreferred range for intrathecal administration of an agent, such as theL-H_(N) (derived from botulinum toxin type A)-substance P, so as toachieve an antinociceptive effect in the patient treated is from about 1U/kg to about 10 U/kg. Intrathecal administration of an agent, such asthe L-H_(N) (derived from botulinum toxin type A)-substance P, in thispreferred range can provide dramatic therapeutic success. Furthermore,our experimental work indicates that a dose of about 3 U/kg can providesignificant and long lasting antinociceptive effect without significantside effects for the treatment of inflammatory and neuropathic pain inhuman patients.

[0117] Although intraspinal administration of the agents is preferredfor the treatment of pain, other routes of administration are possible.For example, the agent according to the invention can also be locallyapplied to a peripheral site of pain to alleviate such pain. A specificexample of this is treatment by local application of the agents into ajoint affected by inflammatory pain. Another example is treatment ofmuscular pain by subcutaneous, preferably intramuscular, injection ofthe agents into the location of pain.

[0118] The present invention includes within its scope the use of anyagent which has a long duration antinociceptive effect when appliedcentrally or peripherally into a patient. For example, agents having theclostridial neurotoxin components made by any of the species of thetoxin producing Clostridium bacteria, such as Clostridium botulinum,Clostridium butyricum, Clostridium beratii and Clostridium tetani can beused or adapted for use in the methods of the present invention.Additionally, all of the botulinum serotypes A, B, C, D, E , F and G canbe advantageously used in the practice of the present invention,although type A is the most preferred and type B the least preferred, asexplained above. Practice of the present invention can provide ananalgesic effect, per injection, for 2 months or longer, for example 6months, in humans.

EXAMPLES

[0119] The following examples provide those of ordinary skill in the artwith specific preferred methods to produce the agents, example 1, and totreat pain, examples 2 through 19, within the scope of the presentinvention and are not intended to limit the scope of the invention.

Example 1 Recombinant Production of Agents

[0120] The production of a fusion of L-H_(N) whereof the L chain isderived from botulinum toxin type B and the amine end segment of the Hchain fragment is derived from botulinum toxin type A. The H_(N)fragment of the botulinum toxin type A is produced according to themethod described by Shone C. C., Hambleton, P., and Melling, J. (1987,Eur. J. Biochem. 167, 175-180) and the L chain of botulinum toxin type Baccording to the method of Sathyamoorthy, V. and DasGupta, B. R. (1985,J. Biol. Chem. 260, 10461-10466). The free cysteine on the amine endsegment of the H chain fragment of botulinum toxin type A is thenderivatized by the addition of a ten-fold molar excess of dipyridyldisulphide followed by incubation at 4 degree C. overnight. The excessdipvridyl disulphide and the thiopyridone by product are then removed bydesalting the protein over a PD10 column (Pharmacia) into PBS.

[0121] The derivatized H_(N) is then concentrated to a proteinconcentration in excess of 1 mg/ml before being mixed with an equimolarportion of L chain from botulinum toxin type B (>1 mg/ml in PBS). Afterovernight incubation at room temperature the mixture is separated bysize exclusion chromatography over Superose 6 (Pharmacia), and thefractions analyzed by SDS-PAGE. The chimeric LH_(N) is then availablefor dramatization to produce a targeted conjugate.

[0122] The example described above is purely illustrative of theinvention. In synthesizing the agents, the coupling of the targetingmoieties to the clostridial components, for example the modifiedclostridial neurotoxins or fragments thereof, is achieved via chemicalcoupling using reagents and techniques known to those skilled in theart. Thus, although the examples given use exclusively the PDPH/EDAC andTraut's reagent chemistry any other coupling chemistry capable ofcovalently attaching the targeting moieties of the agents to clostridialneurotoxin components and known to those skilled in the art is coveredby the scope of this application. Similarly it is evident to thoseskilled in the art that either the DNA coding for either the entirecomposition of the agents or fragments of the agents could be readilyconstructed, and when expressed in an appropriate organism, could beused to recombinantly produce the agents or fragments of the agents.Such genetic constructs of the agents of the invention obtained bytechniques known to those skilled in the art are also covered in thescope of this invention.

Example 2 Treatment of Inflammatory Pain by Intrathecal Administrationof an Agent

[0123] A patient, age 45, experiencing acute inflammatory pain istreated by intrathecal administration, for example by spinal tap to thelumbar region, with between about 0.1 U/kg and 30 U/kg, (preferably from20 U to 500 U), of an agent comprising an L-H_(N) (derived frombotulinum toxin type A)-substance P, the particular agent dose and siteof injection, as well as the frequency of agent administrations dependupon a variety of factors within the skill of the treating physician, aspreviously set forth. Within 1-7 days after agent administration thepatient's pain is substantially alleviated. The duration of painreduction is from about 2 to about 6 months.

[0124] The agent can be injected at different spinal levels to treatdifferent dermosomes, that is to treat pain in various body parts.Additionally, a catheter can be percutaneously inserted into theintrathecal space via lumbar puncture at vertebral level L3-4 or L4-5using a Tuohy needle. When CSF flow is discernible a silastic catheteris threaded cephalad using a C-arm for verification of catheterplacement. The catheter can be advanced to different vertebral locationsand/or used at different dose concentrations to treat different types ofpain and/or spasm. Thus, the catheter can be placed within theintrathecal space at the dermasomal level of the pain or spasmexperienced.

Example 3 Treatment of Neuropathic Pain by Intrathecal Administration ofan Agent

[0125] A patient, age 36, experiencing pain of neuropathic origin istreated by intrathecal administration through spinal tap to the lumbarregion of between about 0.1 U/kg and 30 U/kg, (preferably from 20 U to500 U), of an agent comprising an L-H_(N) (derived from botulinum toxintype A)-substance P. Within 1-7 days the pain symptoms are substantiallyalleviated. The duration of pain reduction is from about 2 to about 6months.

Example 4 Treatment of Pain Subsequent to Spinal Cord Injury byIntrathecal Administration of an Agent

[0126] A patient, age 39, experiencing pain subsequent to spinal cordinjury is treated by intrathecal administration, for example by spinaltap or by catheterization, to the spinal cord, such as to the lumbarregion of the spinal cord, with between about 0.1 U/kg and 20 U/kg,(preferably between 20 U to 500 U), of an agent comprising an L-H_(N)(derived from botulinum toxin type A)-substance P, the particular doseand site of injection, as well as the frequency of administrationsdepend upon a variety of factors within the skill of the treatingphysician, as previously set forth. Within 1-7 days after administrationof the agent the patient's pain is substantially alleviated. Theduration of pain reduction is from about 2 to about 6 months.

Example 5 Treatment of Pain Subsequent to Limb Iniurv by IntrathecalAdministration of an Agent

[0127] A patient, age 51, experiencing pain subsequent to injury to hishand, arm, foot or leg is treated by intrathecal administration, forexample by spinal tap or by catheterization, to the spinal cord, such asto the lumbar region of the spinal cord, with between about 0.1 U/kg and20 U/kg, (preferably from 20 U to 500 U), of an agent comprising L-H_(N)(derived from botulinum neurotoxin type A)-substance P, the particulardose and site of injection, as well as the frequency of administrationsdepend upon a variety of factors within the skill of the treatingphysician, as previously set forth. Within 1-7 days after administrationthe patient's pain is substantially alleviated. The duration of painreduction is from about 2 to about 6 months.

Example 6 Treatment of Pain Associated With Cancer By IntrathecalAdministration of an Agent

[0128] A patient, age 63, suffering from pain associated with cancer istreated by intrathecal administration, for example by spinal tap or bycatheterization, to the spinal cord, such as to the lumbar region of thespinal cord, with between about 1 U/kg and 20 U/kg (preferably about 20U to 500 U), of an agent comprising an LH_(N) (derived from botulinumneurotoxin type A)-substance P, the particular dose and site ofinjection, as well as the frequency of administrations depend upon avariety of factors within the skill of the treating physician, aspreviously set forth. Within 1-7 days after administration the patient'spain is substantially alleviated. The duration of pain reduction is fromabout 2 to about 6 months.

Example 7 Treatment of Pain Associated With Diabetes by IntrathecalAdministration of an Agent

[0129] A patient, age 47, suffering from pain associated with diabetesis treated by intrathecal administration, for example by spinal tap orby catheterization, to the spinal cord, such as to the lumbar region ofthe spinal cord, with between about 0.1 U/kg and 30 U/kg, or 1 to 500 U,of an agent comprising an L-H_(N) (derived from a botulinum neurotoxintype A)-substance P, the particular dose and site of injection, as wellas the frequency of administrations depend upon a variety of factorswithin the skill of the treating physician, as previously set forth.Within 1-7 days after administration the patient's pain is substantiallyalleviated. The duration of pain reduction is from about 2 to about 6months.

Example 8 Treatment of Pain Subsequent to Limb Injury by PeripheralAdministration of an Agent

[0130] A patient, age 35, experiencing pain subsequent to injury to hishand, arm, foot or leg is treated by intramuscular injection withbetween about 1 U/kg and 20 U/kg (preferably from 20 U to 500 U), of anagent comprising L-H_(N) (derived from a botulinum neurotoxin typeA)-substance P. The particular dose and site of injection, as well asthe frequency of administrations depend upon a variety of factors withinthe skill of the treating physician, as previously set forth. Within 1-7days after administration the patient's pain is substantiallyalleviated. The duration of pain reduction is from about 2 to about 6months.

Example 9 Methods for Determining Potency of Botulinum ToxinComponent-Targeting Moiety Conjugates

[0131] The traditional unit of measure for botulinum toxin potency isthe mouse LD₅₀ unit. That is, one unit (1 U) of botulinum toxin is theamount that kills 50% of a group of 18-20 gram female Swiss-Webstermice.

[0132] The unit of measure for potency of a botulinum toxincomponent-targeting moiety conjugate may also be determined by LD₅₀assays. In particular, I U of the botulinum toxin component-targetingmoiety conjugate (e.g., LH_(N)-SP) is the amount of the conjugate thatkills 50% of a group of 18-20 gram female Swiss-Webster mice.

[0133] Alternatively, potency of botulinum toxin component-targetingmoiety conjugate may be determined by the amount of pain reduction in apatient induced by a measured amount of conjugate. For example, the painreduction in a patient may be estimated to be 50% upon intraspinalinjection of a measured amount of conjugate. Thus, the potency can bemeasured as the amount of conjugate that reduces a patient's pain by50%.

[0134] Methods for assessing or quantifying the amount of painexperienced by a subject are well known to those skilled in the art. Forexample, a subject can be given a pain assessment test in which thesubject quantifies the degree of pain based on a scale. One examplewould be assigning the subject's pain a number based on a scale of 1 to10, where a “10” would indicate the worst degree of pain the subjectmight imagine. A pain measure of 4 from an original pain score of 8would be a 50% reduction in pain. Thus, the amount of conjugate requiredto achieve that 50% reduction in pain could be considered 1 U of thebotulinum toxin component-targeting moiety conjugate. Alternatively, thesubject's pain may be measured as the duration of pain. One unit of theconjugate of the invention would accordingly reduce the duration of painby 50%. In addition, a number of physiological measures, such as heartrate, respiratory rate, blood pressure, and diaphoresis, may be usedalone or together with the subjective methods described above, toquantify the amount of the subject's pain.

Example 10 Reduction of Inflammatory Pain by Intrathecal Administrationof an Agent

[0135] A patient, age 45, experiencing acute inflammatory pain istreated by intrathecal administration, for example by spinal tap to thelumbar region, with an amount of an agent comprising an L-H_(N) (derivedfrom botulinum toxin type A)-substance P that reduces pain in thesubject by about 20%, the particular agent dose and site of injection,as well as the frequency of agent administrations depend upon a varietyof factors within the skill of the treating physician, as previously setforth. Within 1-7 days after agent administration the patient's pain isalleviated by about 20% (in particular, the patient's pain score wasoriginally a 5, and after treatment, the patient scored his pain as a4). The duration of pain reduction is from about 2 to about 6 months.

Example 11 Reduction of Inflammatory Pain by Intrathecal Administrationof an Agent

[0136] A patient, age 36, experiencing pain (score of 8) of neuropathicorigin is treated by intrathecal administration through spinal tap tothe lumbar region of an agent comprising an L-H_(N) (derived frombotulinum toxin type A)-substance P that reduces the pain in the subjectby about 50% (score of 4). Within 1-7 days the pain symptoms are reducedby about 50%. The duration of pain reduction is from about 2 to about 6months.

Example 12 Treatment of Osteoid Osteoma With Botulinum Toxin Type A

[0137] A 24 year-old female presents with a four month history of painin the right buttock radiating to the lateral aspect of her thigh andleg. The pain is throbbing in nature and awakens her at night. It isaggravated by exercise and partially alleviated by aspirin. Examinationreveals a full range of hip motion. Routine lab values (hematocrit, WBC,etc.) and CSF content are normal. Pelvic X-rays reveal a small, ovallesion at the base of the right femoral neck. A diagnosis of osteoidosteoma is made. Under radiographic guidance an agent comprising anLH_(N) (derived from botulinum toxin type A)-substance P is injecteddirectly into the tumor. Within 1 to 7 days the pain has been alleviatedby about 80%. Radiography and bone aspiration biopsy at 3 months postinjection fails to reveal any evidence of the neoplasm.

Example 13 Treatment of Osteoid Osteoma With Botulinum Toxin Type B

[0138] A 13 year-old boy is admitted with a three month history ofgnawing, persistent pain in his left thigh. The pain is more pronouncedat night. Both the boy and his parents deny trauma. Physical examinationreveals a healthy boy in no acute distress. Both hip joints have a fullrange of motion. The left thigh is tender. The left patellar reflex isabsent and the ankle jerk somewhat diminished. Plantar responses areboth flexor. Routine lab values, electromyography, spinal fluid contentand pantopaque myelography are all normal. X-rays reveal a small, oval,lytic lesion situated below the lesser trochanter. A diagnosis ofosteoid osteoma is made. An agent comprising an LH_(N) (derived frombotulinum toxin type B)-substance P preparation is injected directlyinto the tumor. Within 1 to 7 days the pain has been reduced by about20%. Radiography and bone aspiration biopsy at 3 months post injectionfails to reveal any evidence of the neoplasm.

Example 14 Treatment of Osteoid Osteoma With Botulinum Toxin Type C₁

[0139] A 58 year-old female is diagnosed with osteoid osteoma. An agentcomprising an LH_(N) (derived from botulinum toxin type C₁)-substance Pis injected directly into the tumor. Within 1 to 7 days the pain hasbeen alleviated and the patient remains asymptomatic. Radiography andbone aspiration biopsy at 3 months post injection fails to reveal anyevidence of the neoplasm.

Example 15 Treatment of Osteoid Osteoma With Botulinum Toxin Type D

[0140] A 56 year-old obese female is diagnosed with osteoid osteoma. Anagent comprising an LH_(N) (derived from botulinum toxin typeD)-substance P is injected directly into the tumor. Within 1 to 7 daysthe pain has been alleviated by about 50% and the patient remainsasymptomatic. Radiography and bone aspiration biopsy at 3 months postinjection fails to reveal any evidence of the neoplasm.

Example 16 Treatment of Osteoid Osteoma With Botulinum Toxin Type E

[0141] A 61 year-old female is diagnosed with osteoid osteoma. An agentcomprising an LH_(N) (derived from botulinum toxin type E)-substance Pis injected directly into the tumor. Within 1 to 7 days the pain hasbeen alleviated by about 20%. Radiography and bone aspiration biopsy at3 months post injection fails to reveal any evidence of the neoplasm.

Example 17 Treatment of Osteoid Osteoma With Botulinum Toxin Type F

[0142] A 52 year-old male is diagnosed with osteoid osteoma. An agentcomprising an LH_(N) (derived from botulinum toxin type F)-substance Pis injected directly into the tumor. Within 1 to 7 days the pain hasbeen alleviated by about 80% and the patient remains asymptomatic.Radiography and bone aspiration biopsy at 3 months post injection failsto reveal any evidence of the neoplasm.

Example 18 Treatment of Osteoid Osteoma With Botulinum Toxin Type G

[0143] A 14 year-old male is diagnosed with osteoid osteoma. An agentcomprising an LH_(N) (derived from botulinum toxin type G)-substance Pis injected directly into the tumor. Within 1 to 7 days the pain hasbeen alleviated by 80% and the patient remains asymptomatic. Radiographyand bone aspiration biopsy at 3 months post injection fails to revealany evidence of the neoplasm.

Example 19 Treatment of Osteoblastoma With Botulinum Toxin Type A-G

[0144] A 19 year old male presents with a two month history ofpersistent pain in the right shoulder Examination reveals a full rangeof shoulder motion. Routine lab values (hematocrit, WBC, etc) and CSFcontent are normal. X-rays reveal a small, oval lesion at the base ofthe scapula and exploratory biopsy confirms a diagnosis ofosteoblastoma. Under radiographic guidance an agent comprising an LH_(N)(derived from botulinum toxin type A, B, C₁, D, E, F, or G)-substance Pis injected directly into the tumor. Within 1 to 7 days the pain hasbeen reduced by about 50% and the patient remains asymptomatic.Radiography and bone aspiration biopsy at 3 months post injection failsto reveal any evidence of the neoplasm.

[0145] Although the present invention has been described in detail withregard to certain preferred methods, other embodiments, versions, andmodifications within the scope of the present invention are possible.For example, a wide variety of neurotoxins can be effectively used inthe methods of the present invention in place of clostridialneurotoxins. Additionally, the present invention includes intraspinaladministration methods wherein two or more agents, such as two or moreagents comprising different clostridial toxin components and targetingmoieties, are administered concurrently or consecutively. For example,an agent comprising a an LH_(N) (botulinum neurotoxin type A)-substanceP can be administered intraspinally until a loss of clinical response orneutralizing antibodies develop, followed by administration of an agentcomprising L-H_(N) (derived from a botulinum neurotoxin typeE)-substance P. While this invention has been described with respect tovarious specific examples and embodiments, it is to be understood thatthe invention is not limited thereto and that it can be variouslypracticed with the scope of the following claims.

1 18 1 11 PRT Unknown Description of Unknown Organism This is asubstance P and is very well known in the art. 1 Arg Pro Lys Pro Gln GlnPhe Phe Gly Leu Xaa 1 5 10 2 12 PRT Unknown Description of UnknownOrganism Precursor to substance P, which is very well known in the art.2 Arg Pro Lys Pro Gln Gln Phe Phe Gly Leu Met Gly 1 5 10 3 13 PRTUnknown Description of Unknown Organism This is a precursor to substanceP and is very well known in the art. 3 Arg Pro Lys Pro Gln Gln Phe PheGly Leu Met Gly Lys 1 5 10 4 14 PRT Unknown Description of UnknownOrganism This is a precursor to substance P and is very well known inthe art. 4 Arg Pro Lys Pro Gln Gln Phe Phe Gly Leu Met Gly Lys Arg 1 510 5 12 PRT Artificial Sequence Description of Artificial Sequence Thisis a carboxy-ester synthetic precursor to substance P. 5 Arg Pro Lys ProGln Gln Phe Phe Gly Leu Met Xaa 1 5 10 6 13 PRT Artificial SequenceDescription of Artificial Sequence This is a carboxy-ester syntheticprecursor to substance P. 6 Arg Pro Lys Pro Gln Gln Phe Phe Gly Leu MetGly Xaa 1 5 10 7 14 PRT Artificial Sequence Description of ArtificialSequence This is a carboxy-ester synthetic precursor to substance P. 7Arg Pro Lys Pro Gln Gln Phe Phe Gly Leu Met Gly Lys Xaa 1 5 10 8 12 PRTArtificial Sequence Description of Artificial Sequence This is acarboxy-ester synthetic precursor to substance P. 8 Arg Pro Lys Pro GlnGln Phe Phe Gly Leu Met Xaa 1 5 10 9 13 PRT Artificial SequenceDescription of Artificial Sequence This is a carboxy-ester syntheticprecursor to substance P. 9 Arg Pro Lys Pro Gln Gln Phe Phe Gly Leu MetGly Xaa 1 5 10 10 14 PRT Artificial Sequence Description of ArtificialSequence This is a carboxy-ester synthetic precursor to substance P. 10Arg Pro Lys Pro Gln Gln Phe Phe Gly Leu Met Gly Lys Xaa 1 5 10 11 4 PRTUnknown Description of Unknown Organism This is a naturally occuringamino thermal peptide fragment derived from substance P. 11 Arg Pro LysPro 1 12 7 PRT Unknown Description of Unknown Organism This is anaturally occuring amino acid thermal peptide fragment derived fromsubstance P. 12 Arg Pro Lys Pro Gln Gln Phe 1 5 13 9 PRT UnknownDescription of Unknown Organism This is a naturally occuring aminothermal peptide frament derived from substance P. 13 Arg Pro Lys Pro GlnGln Phe Phe Gly 1 5 14 11 PRT Artificial Sequence Description ofArtificial Sequence This is an analog of substance P. 14 Arg Xaa Lys ProGln Gln Xaa Phe Xaa Leu Xaa 1 5 10 15 12 PRT Artificial SequenceDescription of Artificial Sequence This is an analog of substance P. 15Arg Xaa Lys Pro Gln Gln Xaa Phe Xaa Leu Met Gly 1 5 10 16 11 PRTArtificial Sequence Description of Artificial Sequence This is an analogof substance P. 16 Arg Xaa Lys Pro Gln Gln Xaa Phe Xaa Leu Xaa 1 5 10 1712 PRT Artificial Sequence Description of Artificial Sequence This is ananalog of substance P. 17 Arg Xaa Lys Pro Gln Gln Xaa Phe Xaa Leu MetGly 1 5 10 18 11 PRT Artificial Sequence Description of ArtificialSequence This is an analog of substance P. 18 Arg Pro Cys Pro Gln CysPhe Tyr Gly Pro Xaa 1 5 10

I claim:
 1. A method for treating pain in a subject, comprisingadministering an agent to the subject, wherein the agent comprises abotulinum toxin component covalently coupled to substance P, therebytreating pain in the subject.
 2. The method of claim 1, wherein thebotulinum toxin component comprises the proteolytic domain of thebotulinum toxin.
 3. The method of claim 1, wherein the botulinum toxincomponent comprises the proteolytic domain and translocational domain ofthe botulinum toxin.
 4. The method of claim 3, wherein the proteolyticdomain is covalently coupled to the translocational domain.
 5. A methodfor reducing pain in a subject, comprising administering an agent to asubject, wherein the agent comprises a botulinum toxin proteolyticdomain covalently attached to a botulinum toxin translocational domain,and substance P covalently attached to the translocational domain,thereby reducing pain in the subject.
 6. The method of claim 5, whereinthe agent is administered intramuscularly.
 7. The method of claim 5,wherein the agent is administered intrathecally.
 8. The method of claim5, wherein the proteolytic domain is a botulinum toxin type Aproteolytic domain.
 9. The method of claim 5, wherein thetranslocational domain is a botulinum toxin type A translocationaldomain.
 10. The method of claim 5, wherein the proteolytic andtranslocational domains are botulinum toxin type A proteolytic andtranslocational domains.
 11. The method of claim 5, wherein the agent isadministered before the onset of a nociceptive event or syndromeexperienced by the subject.
 12. The method of claim 5, wherein the agentis administered after the onset of a nociceptive event experienced bythe subject.
 13. The method of claim 12, wherein the nociceptive eventis a neuropathic pain syndrome.
 14. The method of claim 12, wherein thenociceptive event is inflammatory pain.
 15. A method for reducing painin a subject, comprising administering an agent to the subject, whereinthe agent comprises a botulinum toxin type A proteolytic domaincovalently attached to a botulinum toxin type A translocational domain,and substance P covalently attached to the translocational domain,thereby reducing pain in the subject.
 16. A method for reducing pain ina subject, comprising administering an agent to the subject, wherein theagent comprises a botulinum toxin component coupled to substance P,thereby reducing pain in the subject.
 17. A method for reducing pain ina subject, comprising administering an agent to the subject, wherein theagent comprises a botulinum toxin component coupled to a precursor ofsubstance P, thereby reducing pain in the subject.
 18. A method forreducing pain in a subject, comprising administering an agent to thesubject, wherein the agent comprises a botulinum toxin component coupledto a substance P analogue, thereby reducing pain in the subject.
 19. Themethod of claim 16, wherein the botulinum toxin component is selectedfrom the group consisting of serotype A, serotype B, serotype C₁,serotype D, serotype E, serotype F, and serotype G.
 20. The method ofclaim 16, wherein the botulinum toxin component is botulinum toxinserotype A.
 21. The method of claim 16, wherein the botulinum toxincomponent comprises an H_(N) and an L chain of the botulinum toxin. 22.The method of claim 21, wherein the H_(N) is obtained from a botulinumtoxin selected from the group consisting of botulinum toxin serotype A,serotype B, serotype C₁, serotype D, serotype E, serotype F, andserotype G.
 23. The method of claim 16, wherein the agent comprises anamount of botulinum toxin component that will reduce pain in a patientby about 20%.
 24. The method of claim 16, wherein the agent comprises anamount of botulinum toxin component that will reduce pain in a patientby about 50%.
 25. The method of claim 16, wherein the agent comprises anamount of botulinum toxin component that will reduce pain in a patientby about 80%.
 26. The method of claim 1, wherein the pain is bone tumorpain.
 27. The method of claim 5, wherein the pain is bone tumor pain.